Populations of organisms routinely face abiotic selection pressures, and a central goal of evolutionary biology is to understand the mechanistic underpinnings of adaptive phenotypes. Ultraviolet radiation (UVR) is one of earth’s most pervasive environmental stressors, potentially damaging DNA in any organism exposed to solar radiation. We explored mechanisms underlying differential survival following UVR exposure in genotypes of the water flea Daphnia melanica derived from natural ponds of differing in UVR intensity. The UVR tolerance of a D. melanica genotype from a high-UVR habitat depended on the presence of visible and UV-A light wavelengths necessary for photoenzymatic repair of DNA damage, a repair pathway widely shared across the tree of life. We then measured the acquisition and repair of cyclobutane pyrimidine dimers, the primary form of UVR-caused DNA damage, in D. melanica DNA following experimental UVR exposure. We demonstrate that genotypes from high-UVR habitats repair DNA damage faster than genotypes from low-UVR habitats in the presence of visible and UV-A radiation necessary for photoenzymatic repair, but not in dark treatments. Because differences in repair rate only occurred in the presence of visible and UV-A radiation, we conclude that differing rates of DNA repair, and therefore differential UVR tolerance, are a consequence of variation in photoenzymatic repair efficiency. We then rule out a simple gene expression hypothesis for the molecular basis of differing repair efficiency, as expression of the CPD photolyase gene photorepair did not differ among D. melanica lineages, both in the presence and absence of UVR.